Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches due to the stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis and interactions between Very Late Antigen-4 (VLA-4 or a4b1integrin) and its ligand, Vascular Adhesion Molecule-1 (VCAM-1 or CD106). While HSPCs express CXCR4 and VLA-4, their corresponding ligands, SDF-1 and VCAM-1, are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon acquired hemolytic anemia that results from the expansion of hematopoietic stem cells with a mutation in one of the enzymes (PIG-A) responsible for glycosylphosphatidylinositol (GPI anchor) biosynthesis, which is a post-translation modification of proteins associated with lipid rafts on the cell membrane surface. Some of these proteins are involved in the resistance of erythrocytes to lysis by the final product of complement cascade (CC) activation, C5b-C9, also known as the membrane attack complex (MAC). Furthermore, as we reported, the CXCR4 receptor, which binds a-chemokine stromal derived factor-1 (SDF-1) is also associated with lipid rafts (Blood 2005;105:40–8) similarly as VLA-4 (a4b1integrin). Recently we demonstrated that the bioactive lipid sphingosine-1-phosphate (S1P), which is a major chemoattractant directing egress of HSPCs from bone marrow (BM) into peripheral blood (PB) during mobilization, is released from lyzed erythrocytes in C5b-C9/MAC-dependent manner (Leukemia 2010;24:976–85). We also demonstrated that PNH affected HSPCs are preferentially mobilized into PB which suggests their defective retention in BM microenvironment (Leukemia 2012;26:1722).
To explain preferential mobilization of PNH patients HSPCs into PB we hypothesized that BM residing HSPCs may have a defective adhesion in BM niches due to defect in lipid raft formation that are required for a proper function of CXCR4-SDF-1 and VLA-4-VCAM-1 retention axes.
To address this issue we employed for adhesion, chemotaxis and lipid raft formation assays PIG-A mutated human K-562 and TF-1 cells and more importantly BM MNC derived from PNH patients (n=3). PNH affected HSPCs were sorted by FACS as CD34+FLAER− and normal unaffected HSPCs as CD34+FLAER+cell population. Using these cells we studied their i) adhesiveness to immobilized SDF-1 (CXCR4 ligand), VCAM-1 (VLA-4 ligand), fibronectin and BM stroma cells, ii) chemotactic responsiveness in Transwell assays to SDF-1 gradient, iii) ability to incorporate CXCR4 and VLA-4 receptors into membrane lipid rafts and iv) activation of signaling pathways relevant to cell adhesion/migration (MAPKp42/44 and AKT) in response to SDF-1 stimulation.
We found that both PIG-A mutated human K-562 and TF-1 cells and more important CD34+FLAER− HSPCs derived from BM of PNH patients in contrast to normal CD34+FLAER+HSPCs show defective adhesion to immobilized SDF-1, VCAM-I, fibronectin and BM stroma cells. This corresponded with weak responsiveness of these cells in chemotactic assays to SDF-1 gradient and weaker phosphorylation of MAPK042/44 and AKT after SDF-1 stimulation. Finally, direct confocal microscopy studies demonstrated defect in incorporation of CXCR4 and VLA-4 into membrane lipid rafts in CD34+FLAER− cells isolated from BM of PNH patients.
Based on these observations, we propose a novel view of the pathogenesis of PNH and the expansion of PNH-affected cells in BM microenvironment. Accordingly, the lack of PIG-A protein, which plays an important role in lipid raft formation a proper function of CXCR4 and VLA-4, confers an adhesion defect and an advantage to PNH-affected HSPCs, which become more mobile. These PNH-mutated HSPCs over time may outcompete normal HSPCs for their niches in BM, due to their increased motility, and contribute to the PNH type of hematopoiesis. Defective retention of PNH affected HSPCs and they proper interaction with stem cell niches in BM may lead with time in some of the cases to their malignant transformation. We propose also that drugs that increase BM level of S1P and thus prevent egress of PNH HSPCs from their niches could be employed in PNH treatment along with CC inhibitors (Eculizumab).
Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.
Asterisk with author names denotes non-ASH members.